The first developmental choice faced by germ cells in the majority of higher eukaryotes is oogenesis versus spermatogenesis, but very little is known about how this basic event is genetically encoded in any organism. We have chosen to study the developmental genetics of this germline sex determination process in the fruit fly, Drosophila melanoaster. Our long-term goals are to: 1) identify the major germline sex determination regulatory genes, 2) determine how they are related in terms of a developmental hierarchy and 3) understand in detail how these regulatory gene products function. We are question oriented and take advantage of genetic, molecular, and biochemical techniques as needed. Current molecular studies are focused on OVO, a highly conserved zinc finger domain protein functioning at a key intersection in the hierarchy. We have found that the ovo+ locus has alternate promotors and 5' exons, only one of which has an in frame AUG. One of these isoforms is produced only in females and is controlled by the sex-karyotype and somatic inductive signals. We have sequenced dominant negative mutations in the locus and find that they add new in frame AUGs downstream of the exon that bears no AUG codon of its own. The effect of these mutations is to change the female-specific OVO isoform into a non-sex specific form. Newly engineered AUGs also result in dominant negative phenotypes and introduced stop codons abolish the dominant negative activity. We are also looking for biochemical function by assaying for the binding of bacterially expressed OVO to its own promotor and to the ovarian tumor promoter (we have shown that both are regulated by ovo+ dose). Interestingly OVO binds at the transcription initiation site of both these TATA-less promoters. We have also developed antibodies to OVO, which we are testing in situ for localization patterns, in western blots to detect different isoforms.